1. Field of the Invention
The present invention relates to a device and a method for fusing biological cells in a cell suspension, more particularly to a chamber for fusing cells without using dielectrophoresis and to a method of fusing cells by electroporation without using a dc pulse.
2. Description of the Related Art
Cell fusion is useful for the production of monoclonal antibodies or giant cells, or the introduction of a high molecular weight substance into a cell.
Conventionally, a method of cell fusion by chemical adhesion using polyethylene glycol (PEG) has been frequently used. However, since PEG is toxic to cells, fusion yields will be dependent on the quality of PEG. With most PEG, the ratio of formed hybridoma to lymphocytes is 1:2.times.10.sup.5, and with the most effective PEG, the ratio is 1:5.times.10.sup.4 (S. Fazekas de St. Grouth and D. Scheidegger, J. Immunol, Method, 35, 1, 1980).
In recent years, electroporation, a method of applying a high voltage pulse to make pores in cell membranes, has been used for cell fusion or the introduction of a gene. The cell fusion method using electroporation is called electrofusion, electro-cell-fusion, etc. This method includes a first step of contiguously aligning cells in a cell solution between a pair of electrodes, and a second step of applying a high voltage pulse to the contiguously aligned cells.
U.S. Pat. No. 4,441,972 discloses a cell fusion method using a chamber defined by two parallel upper and lower electrode plates. In the first step, dielectrophoresis is performed with a weak ac voltage (1 to 20 volts, 200 to 600 kHz) to form pearl chains of cells between the parallel electrode plates. Then, in the second step, electroporation is carried out with a brief dc pulse (10 to 250 volts, 1 to 200 microsec).
European Patent Application, Publication No. 193769 discloses a method using a reaction tube applied to a centrifuge. In the first step, precipitated cell layers are formed at the bottom of the reaction tube by centrifugation. In the second step, a pair of electrode are inserted in the reaction tube, and electroporation is effectuated with a short dc pulse (2.5 to 5.0 kV/cm, 30 microsec).
In commonly used cell fusion apparatus, the ac voltage used for dielectrophoresis is in the range of 40 to 200 V.sub.p-p and 0.25 to 20 MHz, and the dc voltage used for electroporation is in the range of 200 to 3,000 V and 0.1 microsec to 100 ms. However, the method using dielectrophoresis requires a two-step treatment with ac and dc power so that its construction is complicated. On the other hand, in the method using centrifugation, it is difficult to reproduce the optimum arrangement between the cells and the electrodes because the electrodes are inserted after centrifugation. Further, in the electroporation using a dc pulse, the reproducibility of the voltage condition is low because of an unstable pulse wave having overshoot or ringing, so that a high-level control means is necessary.